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Cited by 12Ovarian cancer diagnosis and treatmentGlycosylation and Glycoproteins ResearchCarbohydrate Chemistry and Synthesis

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A Revised Molecular Model of Ovarian Cancer Biomarker CA125 (MUC16) Enabled by Long-read Sequencing

Open access
Wang, Chien-Wei, Weaver, Simon D., Boonpattrawong, Nicha, Schuster-Little, Naviya, Patankar, Manish, Whelan, Rebecca J.·2024·Cancer Research Communications
DOI 10.1158/2767-9764.crc-23-0327PMC PMC10829539

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Abstract

The biomarker CA125, a peptide epitope located in several tandem repeats of the mucin MUC16, is the gold standard for monitoring regression and recurrence of high-grade serous ovarian cancer in response to therapy. However, the CA125 epitope along with several structural features of the MUC16 molecule are ill defined. One central aspect still unresolved is the number of tandem repeats in MUC16 and how many of these repeats contain the CA125 epitope. Studies from the early 2000s assembled short DNA reads to estimate that MUC16 contained 63 repeats. Here, we conduct Nanopore long-read sequencing of MUC16 transcripts from three primary ovarian tumors and established cell lines (OVCAR3, OVCAR5, and Kuramochi) for a more exhaustive and accurate estimation and sequencing of the MUC16 tandem repeats. The consensus sequence derived from these six sources was confirmed by proteomics validation and agrees with recent additions to the NCBI database. We propose a model of MUC16 containing 19—not 63—tandem repeats. In addition, we predict the structure of the tandem repeat domain using the deep learning algorithm, AlphaFold. The predicted structure displays an SEA domain and unstructured linker region rich in proline, serine, and threonine residues in all 19 tandem repeats. These studies now pave the way for a detailed characterization of the CA125 epitope. Sequencing and modeling of the MUC16 tandem repeats along with their glycoproteomic characterization, currently underway in our laboratories, will help identify novel epitopes in the MUC16 molecule that improve on the sensitivity and clinical utility of the current CA125 assay. Significance: Despite its crucial role in clinical management of ovarian cancer, the exact molecular sequence and structure of the biomarker, CA125, are not defined. Here, we combine long-read sequencing, mass spectrometry, and in silico modeling to provide the foundational dataset for a more complete characterization of the CA125 epitope.

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Figures

FIGURE 1 Gel image of cDNA products from RT-PCR. Lane 1: NEB 1 kb Extend DNA Ladder. Lanes 2–4: RT-PCR product from Kuramochi, OVCAR3 and OVCAR5 cells, respectively. The asterisk indicates the location of the approximate 10 kbp cDNA product.
FIGURE 1 Gel image of cDNA products from RT-PCR. Lane 1: NEB 1 kb Extend DNA Ladder. Lanes 2–4: RT-PCR product from Kuramochi, OVCAR3 and OVCAR5 cells, respectively. The asterisk indicates the location of the approximate 10 kbp cDNA product.CC BY · PubMed Central
FIGURE 2 Proteomic analysis of immunoprecipitated MUC16. Identified MUC16 peptides from immunoprecipitated (A) OVCAR3 cell lysate and (B) pooled ascites mapped onto the proposed repeat domains of MUC16. The repeat domains are each represented by a horizontal line, and the amino acid position within the repeat is on the x-axis. Repeats shown in green (8--12) are the five repeats not included in the NM_024690.2 sequence (MUC16 isoform 4). Rectangles represent peptides, where the pink peptides are unique to repeats 8–12 (map to nowhere else in MUC16) and blue peptides are not unique to repeats 8–12. All peptides shown are unique to MUC16 (map to nowhere else in the proteome).
FIGURE 2 Proteomic analysis of immunoprecipitated MUC16. Identified MUC16 peptides from immunoprecipitated (A) OVCAR3 cell lysate and (B) pooled ascites mapped onto the proposed repeat domains of MUC16. The repeat domains are each represented by a horizontal line, and the amino acid position within the repeat is on the x-axis. Repeats shown in green (8--12) are the five repeats not included in the NM_024690.2 sequence (MUC16 isoform 4). Rectangles represent peptides, where the pink peptides are unique to repeats 8–12 (map to nowhere else in MUC16) and blue peptides are not unique to repeats 8–12. All peptides shown are unique to MUC16 (map to nowhere else in the proteome).CC BY · PubMed Central
FIGURE 3 Proteomic analysis of immunoprecipitated and patient-derived samples. Identified MUC16 peptides from immunoprecipitated (A) OVCAR3 cell lysate and (B) pooled ascites mapped onto the 63 tandem repeat model. Identified MUC16 peptides from patient-derived ascites (C) Patient 1, (D) Patient 2, and (E) Patient 3 mapped onto the 63 tandem repeat model. The repeat domains are each represented by a horizontal line, and the amino acid position within the repeat is on the x-axis. Repeats shown in green (6, 10, 12--53) are the 44 repeats in the 63 tandem repeat model, but not our 19 tandem repeat model. No peptides are identified to be unique to repeats 6, 10, 12–53. All peptides shown are unique to MUC16 (map to nowhere else in the proteome).
FIGURE 3 Proteomic analysis of immunoprecipitated and patient-derived samples. Identified MUC16 peptides from immunoprecipitated (A) OVCAR3 cell lysate and (B) pooled ascites mapped onto the 63 tandem repeat model. Identified MUC16 peptides from patient-derived ascites (C) Patient 1, (D) Patient 2, and (E) Patient 3 mapped onto the 63 tandem repeat model. The repeat domains are each represented by a horizontal line, and the amino acid position within the repeat is on the x-axis. Repeats shown in green (6, 10, 12--53) are the 44 repeats in the 63 tandem repeat model, but not our 19 tandem repeat model. No peptides are identified to be unique to repeats 6, 10, 12–53. All peptides shown are unique to MUC16 (map to nowhere else in the proteome).CC BY · PubMed Central
FIGURE 4 Schematic models of MUC16. Top, The model derived from the sequence determined by O'Brien and colleagues (2001) containing 63 units in the tandem repeat domain. Bottom, The model proposed in this study, containing 19 units in the tandem repeat domain. The repeats that are not included in the proposed model (6, 10, and 12 through 53) are shown as white circles. Ellipses represent repeats that are not shown for clarity. The N-terminal domain is much larger than shown here and is also compressed for clarity.
FIGURE 4 Schematic models of MUC16. Top, The model derived from the sequence determined by O'Brien and colleagues (2001) containing 63 units in the tandem repeat domain. Bottom, The model proposed in this study, containing 19 units in the tandem repeat domain. The repeats that are not included in the proposed model (6, 10, and 12 through 53) are shown as white circles. Ellipses represent repeats that are not shown for clarity. The N-terminal domain is much larger than shown here and is also compressed for clarity.CC BY · PubMed Central
FIGURE 5 MUC16 model proposed in this study. The model includes: a highly glycosylated N-terminal domain, a 19-unit tandem repeat domain that contains the CA125 epitopes, and a short C-terminal domain that includes a membrane-spanning region. The inset shows a representative 3D structure of one tandem repeat domain, predicted by AlphaFold. The C-loop (residues 59–79) is shown in red, with the cysteine residues shown in salmon. The unstructured proline/serine/threonine-rich region is omitted for clarity. White slashes within the N-terminal domain and tandem repeat domain show locations where amino acids are not explicitly shown for clarity. Figure created with https://Biorender.com.
FIGURE 5 MUC16 model proposed in this study. The model includes: a highly glycosylated N-terminal domain, a 19-unit tandem repeat domain that contains the CA125 epitopes, and a short C-terminal domain that includes a membrane-spanning region. The inset shows a representative 3D structure of one tandem repeat domain, predicted by AlphaFold. The C-loop (residues 59–79) is shown in red, with the cysteine residues shown in salmon. The unstructured proline/serine/threonine-rich region is omitted for clarity. White slashes within the N-terminal domain and tandem repeat domain show locations where amino acids are not explicitly shown for clarity. Figure created with https://Biorender.com.CC BY · PubMed Central
FIGURE 6 AlphaFold predicted MUC16 tandem repeat model. The 19 tandem repeat structures were predicted individually and overlaid. All repeats contain a similar structure including two alpha helices, one beta sheet, and a proline/serine/threonine-rich disordered region (this region is not predicted with high confidence and is omitted for clarity).
FIGURE 6 AlphaFold predicted MUC16 tandem repeat model. The 19 tandem repeat structures were predicted individually and overlaid. All repeats contain a similar structure including two alpha helices, one beta sheet, and a proline/serine/threonine-rich disordered region (this region is not predicted with high confidence and is omitted for clarity).CC BY · PubMed Central
FIGURE 7 Venn diagram comparing peptides from two MUC16 models. An in silico tryptic digest was done of the 63 and 19 tandem repeat models of MUC16. The Venn diagram compares the peptides from the two models that would be detectable by mass spectrometry.
FIGURE 7 Venn diagram comparing peptides from two MUC16 models. An in silico tryptic digest was done of the 63 and 19 tandem repeat models of MUC16. The Venn diagram compares the peptides from the two models that would be detectable by mass spectrometry.CC BY · PubMed Central

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Metadata

Authors
Wang, C.-W., Weaver, S. D., Boonpattrawong, N., Schuster-Little, N., Patankar, M., & Whelan, R. J.
Affiliations
  • University of Kansas (US)
  • University of Notre Dame (US)
  • University of Wisconsin–Madison (US)
Corresponding author
Boonpattrawong, Nicha
ORCID
Venue
Cancer Research Communications
Publisher
American Association for Cancer Research (AACR)
Published
2024-01-31
Volume
4
Issue
1
Pages
253-263
ISSN
2767-9764 (ISSN-L)
DOI
10.1158/2767-9764.crc-23-0327
PMC
PMC10829539
OpenAlex
W4390703493
Cited by
12
References
51
Citations by year
2026: 3 · 2025: 6 · 2024: 3
FWCI
4.73
Type
journal article
Language
en
Open access
Licence
Locations
  • Cancer Research Communications (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Funders
  • HHS | NIH | National Cancer Institute (R21CA267532)
  • Tell Every Amazing Lady About Ovarian Cancer Louisa M. McGregor Ovarian Cancer Foundation (n/a)
  • HHS | NIH | National Institute of General Medical Sciences (T32GM075762)
  • Veterans Administration Medical Center (5I01BX005627)
  • Diane Lindstrom Funds (n/a)
Topics
  • Health Sciences › Medicine › Oncology › Ovarian cancer diagnosis and treatment
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Glycosylation and Glycoproteins Research
  • Physical Sciences › Chemistry › Organic Chemistry › Carbohydrate Chemistry and Synthesis
Keywords
CA125, ovarian cancer biomarker, tandem repeats, long-read sequencing, nanopore sequencing, AlphaFold structure prediction, SEA domain, mucin structure, high-grade serous ovarian cancer, glycoproteomics, OVCAR3
MeSH terms
Epitopes, Female, Humans, Membrane Proteins, Models, Molecular, Ovarian Neoplasms, Biomarkers, Tumor, Apoptosis, CA-125 Antigen, Cell Line, Tumor

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